Regulation and Selectivity of Exchange Factors for G-proteins of the Ras-family

Small G-proteins are important regulators of the cellular signaling pathways. Among them, members of the Ras family of small G-proteins regulate processes such as cell differentiation, growth, migration, transport and adhesion, and their deregulation may lead to various diseases. Small G-proteins switch between GDP-bound and GTP-bound conformations, whereas only GTP-bound form can bind to the effector proteins and thereby transduce the biological signal. Guanine nucleotide exchange factors (GEFs) catalyze the activation of small G-proteins by promoting the release of GDP and the binding of GTP. Therefore, studies of GEFs that activate the Ras family members are of high interest. In this thesis we studied several aspects of the function of the Ras family GEFs. We investigated the regulation of C3G, GEF for Rap. C3G has been shown to be involved in embryonic development, control of the formation of adherens junctions and migration. We have demonstrated in vitro that C3G is autoinhibited, and that the autoinhibition is relieved upon the interaction with adaptor protein Crk and tyrosine phosphorylation by Src. We propose a mechanism of activation of C3G in which the Src-catalyzed phosphorylation and interaction with Crk function together to break the interactions established between the inhibitory regions from the N-terminal part of C3G and the catalytic region of C3G. We further studied the function and the structure of Rlf, GEF for Ral. Rlf has been previously shown to interact with the active forms of Rap and Ras, and the interaction with active Ras has been suggested to influence the cellular localization of Rlf. We demonstrate that interaction of Rlf with Ras or Rap does not influence the catalytic activity of Rlf. We also determined the crystal structure of the catalytic module of Rlf, which contains the REM domain and the CDC25-HD. This structure is distinguished by an extended three-stranded β-sheet that precedes the REM domain. The β-sheet element is conserved among the other members of RalGDS family of Ral GEFs. However, Rlf contains a unique proline-rich sequence in the β-sheet, that establishes interactions with the SH3 domains of several Rlf-interacting proteins. β-sheet keeps the proline-rich sequence in the preferential conformation for the interaction with the SH3 domains, and this conformational pre-selection is suggested to be responsible for the gain of affinity in the interactions with the SH3 domains. Finally, we investigated the basis of selectivity of the Ras family GEFs towards different Ras family small G-proteins. Ras and Rap exhibit high sequence homology, but most GEFs are able to distinguish between them based on the small number of non-conserved residues. Using mutational analysis we identify the most critical residues that are responsible for the differential recognition of Ras and Rap. This mutational analysis allows the prediction of the selectivity of the tested GEFs. Furthermore, we systematically analyzed selectivity of 14 members of Ras family of G-proteins and 9 Ras family GEFs. This dataset helps to clarify the assignment of GEFs to G-proteins in the cases that are reported controversially in literature